Assay systems capable of detecting the presence or absence of antibodies generated in response to the presence of antigens are well known. Such assay systems have proved useful in, inter alia, the diagnosis of various diseases. For example, viral infections, such as AIDS (acquired immune deficiency syndrome) and CMV (cytomegalovirus) may be diagnosed with assays which detect the presence of viral antibodies in patients suspected of having the disease. Examples of such assay systems which employ antigen-antibody binding include ELISA, Western Blot, Quick Western Blots (U.S. Pat. Nos. 4,816,387 and 4,885,235) and RIA. Such diagnostics uniformly include controls to insure the integrity of the test system.
Typically, the diagnostics have both positive and negative controls. The positive control provides pertinent information concerning the activitY of the test system, i.e., that reactive antibodies specific to the antigens used in an antibody test system are bound to the antigens (indicating that the antigens used in the test system are working properly), and that the anti-immunoglobulin used to detect the bound immunoglobulin is working. In the case of an ELISA system the anti-immunoglobulin may be labeled with an enzyme (conjugate) which activates a substrate added to the system to give a chromogen reaction; in this case the positive control indicates whether the conjugate has reacted, and whether the substrate has worked properly as an activated chromogen. A negative control provides information about the absence of reactive antibodies specific to the particular antigens used in a test system. It also provides information as to the reaction level, determined by the signal used in a particular test, at which a specimen may be considered negative.
The cut-off point in a particular test is often based upon the relative value obtained by a positive control and/or by the negative control. An acceptable detection range obtained by the controls utilized with a particular type of test kit is specifically designed and titrated for that tYpe of kit. The positive control "value" obtained in a particular test system affects the sensitivity of that test system; the negative control "value" affects the specificity of the test system.
Presently, the antibody used for a positive control is obtained from seropositive human donors. As can be appreciated, it is disadvantageous to depend upon seropositive donors as the sole source of antibody required for a positive control. Notably, the supply of antibodies is scarce and uncertain and the quality and characteristics of the antibody varies from donor to donor. Further, as more successful therapies become known and used, fewer seropositive donors will be available, and thus the required antibody even more difficult to obtain.
In the case of AIDS patients it has been found that the condition of patients who donate blood or are subjected to plasmaphoreisis deteriorates rapidly. Therefore, obtaining AIDS positive blood or plasma from patients can accelerate the disease and accordingly, using AIDS patients as a source of antibody for use as a positive control should be avoided.
The previously mentioned assay systems detect the presence or absence of IgG (immunoglobulin G). Such assays only allow "controlled" detection (measurement defined by use of anti-IgG conjugate and of antibody positive control) of the presence of IgG in blood and body fluids directed to antigens used in the test systems. The appearance of detectable IgG directed to antigens in an infected/immunized individual does not occur until 30-40 days after initial infection in many instances. The IgG class antibodies are often present for months or years after infection/immunization.
The presence of circulating IgG directed to immunizing antigens during the course of an infection (or after immunization) is preceded by the presence of circulating IgM antibodies directed towards the antigens/immunogens. IgM antibodies directed to antigens in an infected/immunized individual are often present in detectable quantities as early as 14 days (or earlier) after the infection/immunization. The IgM class antibodies gradually lose titer 30-35 days after initial infection/immunization.
It is widely recognized that diagnostics which can detect antibodies other than IgG are desirable. For example, it is known that generally after confrontation with a foreign body, the human immune system responds by generating antibodies against the foreign body or antigen. It is believed that IgM, not IgG is produced first. As can be appreciated, assays capable of detecting IgM will facilitate early detection of numerous diseases. IgM is, however, a relatively short-lived antibody. While it may be produced shortly after infection, IgM levels fall, eventually below detectable levels, as IgG is produced, in increasing amounts. See FIG. 1 which compares the titer of IgM and IgG in a pig immunized with HIV-1. Because IgM has a short life span, IgM levels are typically below detectable levels before many diseases are even diagnosed. Therefore, IgM is not readily obtainable from seropositive donors and a dependable, reliable source of this important antibody is needed.
The present invention overcomes the previously mentioned disadvantages because it provides the ability to produce the desired antibody, i.e., IgG and IgM in a non-human species. In accordance with the present invention there is provided a method of using non-human immune antibody as a positive control in assay systems.